Normal keratinocytes proliferate when cultured in medium with 0.02-0.10 mM calcium and terminally differentiate when medium calcium is increased to greater than 0.1 mM. In contrast, neoplastic keratinocyte cell lines maintain the potential for continued cell renewal and survive when external calcium is increased. In order to determine whether elevation of extracellular calcium produced changes in intracellular free calcium (Cai) levels, Cai was measured in individual living keratinocytes by use of the fluorescent calcium probe fura-2. Most normal keratinocytes responded to increased extracellular calcium by a gradual 2- to 3-fold increase in Cai lasting for at least 28 min. A subpopulation displayed a sharp peak of Cai at 2 min. In contrast, the Cai level in neoplastic cells in either low or high calcium medium was 2- to 3-fold higher than that in normal cells, and all cells in the population showed a transient 4- to 9-fold elevation of Cai 2 min after external calcium was increased. Thus normal and neoplastic keratinocytes differ in the level of Cai under low calcium conditions and in their response to elevated external calcium. The regulation of Cai in keratinocytes may be important in determining their potential for terminal differentiation.

Cultured normal murine keratinocytes maintain a basal cell phenotype in medium with a Ca2+ concentration of 0.05 mM and differentiate when exposed for 28-48 h to medium supplemented with extracellular Ca2+ greater than 0.10 mM. Previous studies have documented Ca2+ activation of signaling pathways in the plasma membrane and tightly regulated cellular responses to small incremental changes in extracellular Ca2+. To determine if changes in free cytosolic calcium (Cai) are associated with these early signaling events, digital image analysis of fura-2-loaded keratinocytes was used to measure Cai in individual cells. Basal keratinocytes in 0.05 mM Ca2+ display a biphasic Cai increase when exposed to greater than 0.1 mM Ca2+ in serum-containing medium. These separate phases were controlled by different media components. Initial peak Cai occurred rapidly (within 60 s), was transient (lasting less than 5 min), and resulted from release of 10-20% of total intracellular Ca2+ stores. Peak Cai depended on serum concentration and was independent of extracellular Ca2+. This transient Cai response was lost as keratinocytes differentiated. Plateau Cai level was sustained (greater than 24 h) and depended on extracellular Ca2+, but not serum. The magnitude of plateau Cai increased incrementally following increases in extracellular Ca2+ as small as 0.02 mM. A similar biphasic Cai increase was noted in cultures of murine dermal fibroblasts stimulated by 1.2 mM Ca2+ and serum. However, fibroblasts did not lose the serum response in high-Ca2+ medium, and plateau Cai was not sensitive to small changes in extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

A human corneal epithelial cell line, 10.014 pRSV-T (HCE-T cells), has been used to develop a three-dimensional in vitro model of the human comeal epithelium (HCE-T model). HCE-T cells form a stratified culture when grown at the air-liquid interface on a collagen membrane in serum-free medium. This model served as the basis for assays which supported the ocular irritancy assessment of water-soluble test substances. In vitro assays used were transepithelial permeability to sodium fluorescein (TEP) and transepithelial electrical resistance (TER). These measured alterations in the barrier function of this comeal epithelial equivalent Barrier function is a well-developed property in the HCE-T model that supports the mechanistic relevance of these assays. In vitro data, averaged from replicate assays, were compared to respective Drake rabbit eye irritation data from the publicly available ECETOC and CTFA databases using linear regression with Pearson's correlation analysis. For chemicals, Pearson's correlation coefficients, r, from comparisons of Draize maximum average scores (MAS) to TEP and TER data were 0.71 and 0.55, respectively. For product formulations, Pearson's correlation coefficients from comparisons of Draize MAS to TEP and TER data were 0.86 and 0.80, respectively. Data indicated that barrier function alterations in the HCE-T model correlated with ocular irritancy and comeal toxicity. While the irritancy of the chemicals tested was effectively assessed only by the TEP assay,

The liver-derived human cell line, Hep G2, has high benzo[a]pyrene-metabolizing activity and converts benzo[a]pyrene to intermediates that are mutagenic and that bind to DNA. This cell line will be useful for studying metabolic activation of polycyclic aromatic hydrocarbons and other xenobiotics by human tissue and as an activation system in short-term screening assays for identifying compounds with carcinogenic potential for humans.

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